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Inborn errors of metabolism (IEM) are a highly heterogeneous group of genetic conditions and represent a relevant cause of morbidity and mortality in the pediatric population. IEM, which are individually rare but collectively numerous, are well-recognized entities of the generic class of 'rare' diseases. Since the first descriptions by Garrod at the beginning of the 20th century, several hundred new disorders have been defined, as new biochemical and molecular diagnostic tools became available. The clinical pictures of single diseases are extremely diverse, ranging from acute life-threatening manifestations to chronic late-onset forms, with single or multiorgan involvement. Mental retardation and progressive neurological impairment often characterize the clinical course. One of the principles to prevent high morbidity and mortality rates is early recognition followed by prompt therapeutic intervention. Therefore, a small number of treatable IEM is subject to neonatal mass screening. More recently, an innovative technique, based on tandem mass spectrometry, has expanded the range of neonatal screening to several additional disorders. Owing to the extreme heterogeneity, as well as to the increasing number of new disorders, exhaustive and updated epidemiological data on the overall occurrence of IEM are lacking. A national retrospective study was conducted to define the epidemiological profile of IEM in Italy and to estimate the costs related to the disease burden. Other relevant issues of our investigations focused on creating protocols of treatment for neonatal IEM, and on the development of new methods for the biochemical diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: To evaluate and describe the diagnostic process, medical, nutritional, and surgical approach, and neurological outcome, we report data from a large Italian cohort of patients with congenital hyperinsulinism (CHI)., Methods: We retrospectively analyzed 154 CHI patients admitted to Ospedale Pediatrico Bambino Gesù from 1985 to 2022., Results: Hypoglycemia occurred within the first year of life in 85.5% of patients, median time to diagnosis was 1 day (IQR 14 days). Ninety-two percent of patients were treated with diazoxide: 66.9% were responsive. Octreotide was administered to 28.6% of patients: 61.4% were responsive. Forty percent of patients were off-therapy, mostly from diazoxide. Thirty-four percent of patients carried mutations in ABCC8, 12.6% were syndromic, and 9.2% were transient CHI. Surgery was performed in 23/47 diazoxide-unresponsive and 2/95 diazoxide-responsive patients: 64.0% were focal at histology. Combining data from genetics, pancreatic venous sampling, 18F-DOPA PET/CT, and histology, 80.6% resulted diffuse, 16.7% focal, and 2.8% atypical CHI. Post-surgical diabetes developed in 6 patients. Neurocognitive evaluation revealed developmental delay or intellectual disability in 15.7% of 70 patients, mostly of a mild degree. Epilepsy was documented in 13.7% of 139 patients., Conclusion: Our diagnostic and therapeutic results are mainly consistent with the international indications and the CHI Global Registry data, with relatively low rates of neurological outcomes. Good outcomes were likely associated with early diagnosis and prompt management of patients because the majority of patients were diagnosed within 2 weeks. Remarkably, it is of utmost importance to spread the knowledge and refer CHI patients to multidisciplinary expert centers., (© 2024 S. Karger AG, Basel.)

Multifunctional micromanipulation systems have garnered significant attention due to the growing interest in biological and medical research involving model organisms like zebrafish ( Danio rerio ). Here, we report a novel acoustofluidic rotational micromanipulation system that offers rapid trapping, high-speed rotation, multi-angle imaging, and 3D model reconstruction of zebrafish larvae. An ultrasound-activated oscillatory glass capillary is used to trap and rotate a zebrafish larva. Simulation and experimental results demonstrate that both the vibrating mode and geometric placement of the capillary contribute to the developed polarized vortices along the long axis of the capillary. Given its capacities for easy-to-operate, stable rotation, avoiding overheating, and high-throughput manipulation, our system poses the potential to accelerate zebrafish-directed biomedical research.

Receiving information in the case of a positive or false-positive expanded newborn screening (ENBS) result for metabolic diseases is a stressful event. The availability of psychological support to families is crucial across the different communication steps and is recommended by different guidelines and position papers. However, more information is needed about the availability of psychological resources in the ENBS process. This national survey aimed to provide an overview of the availability of psychological resources for parents who received communication of positivity at the ENBS in the 23 Italian centers and how the support is provided to parents. An online survey was sent to the Heads of the ENBS centers asking about the availability of a clinical psychologist, their involvement in the ENBS process, and an estimation of parents receiving psychological support. More than 60% of the centers report having a clinical psychologist in the ENBS team; however, in more than 50% of cases, the psychologist does not participate in the consultation with parents (nor for the first consultation post-positivity or at confirmation of diagnosis). Furthermore, nearly 60% of the centers reported the experience of parental rejection of psychological sessions.  Conclusion: There is a need for harmonization among the Italian ENBS centers concerning the availability of psychological resources and how these resources are provided to families. Parents' needs remained only partially fulfilled. What is Known: • Receiving communication of positivity at the ENBS can be highly stressful for parents and requires adequate psychological support. • The guidelines recommend psychological support for parents during the ENBS process. What is New: • Only 14/23 (60.9%) of Italian ENBS centers have a clinical psychologist within the team. • In half of the consultations with parents receiving communication of positivity, the clinical psychologist is never involved., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Organic acidurias (OAs), urea-cycle disorders (UCDs), and maple syrup urine disease (MSUD) belong to the category of intoxication-type inborn errors of metabolism (IT-IEM). Liver transplantation (LTx) is increasingly utilized in IT-IEM. However, its impact has been mainly focused on clinical outcome measures and rarely on health-related quality of life (HRQoL). Aim of the study was to investigate the impact of LTx on HrQoL in IT-IEMs. This single center prospective study involved 32 patients (15 OA, 11 UCD, 6 MSUD; median age at LTx 3.0 years, range 0.8-26.0). HRQoL was assessed pre/post transplantation by PedsQL-General Module 4.0 and by MetabQoL 1.0, a specifically designed tool for IT-IEM. PedsQL highlighted significant post-LTx improvements in total and physical functioning in both patients' and parents' scores. According to age at transplantation (≤3 vs. >3 years), younger patients showed higher post-LTx scores on Physical (p = 0.03), Social (p < 0.001), and Total (p =0.007) functioning. MetabQoL confirmed significant post-LTx changes in Total and Physical functioning in both patients and parents scores (p ≤ 0.009). Differently from PedsQL, MetabQoL Mental (patients p = 0.013, parents p = 0.03) and Social scores (patients p = 0.02, parents p = 0.012) were significantly higher post-LTx. Significant improvements (p = 0.001-0.04) were also detected both in self- and proxy-reports for almost all MetabQoL subscales. This study shows the importance of assessing the impact of transplantation on HrQoL, a meaningful outcome reflecting patients' wellbeing. LTx is associated with significant improvements of HrQol in both self- and parent-reports. The comparison between PedsQL-GM and MetabQoL highlighted that MetabQoL demonstrated higher sensitivity in the assessment of disease-specific domains than the generic PedsQL tool., (© 2023 SSIEM.)

Liver and liver/kidney transplantation are increasingly used in methylmalonic aciduria, but little is known on their impact on CNS. The effect of transplantation on neurological outcome was prospectively assessed in six patients pre- and post-transplant by clinical evaluation and by measuring disease biomarkers in plasma and CSF, in combination with psychometric tests and brain MRI studies. Primary (methylmalonic- and methylcitric acid) and secondary biomarkers (glycine and glutamine) significantly improved in plasma, while they remained unchanged in CSF. Differently, biomarkers of mitochondrial dysfunction (lactate, alanine, and related ratios) significantly decreased in CSF. Neurocognitive evaluation documented significant higher post-transplant developmental/cognitive scores and maturation of executive functions corresponding to improvement of brain atrophy, cortical thickness, and white matter maturation indexes at MRI. Three patients presented post-transplantation reversible neurological events, which were differentiated, by means of biochemical and neuroradiological evaluations, into calcineurin inhibitor-induced neurotoxicity and metabolic stroke-like episode. Our study shows that transplantation has a beneficial impact on neurological outcome in methylmalonic aciduria. Early transplantation is recommended due to the high risk of long-term complications, high disease burden, and low quality of life., (© 2023 SSIEM.)

Phenylketonuria (PKU) is an inherited metabolic disease characterized by a defective conversion of phenylalanine (Phe) to tyrosine, potentially leading to Phe accumulation in the brain. Dietary restriction since birth has led to normal cognitive development. However, PKU patients can still develop cognitive or behavioral abnormalities and subtle neurological deficits. Despite the increasing evidence in the field, the assessment of neurocognitive, psychopathological, and neurological follow-up of PKU patients at different ages is still debated. The high interindividual variability in the cognitive outcome of PKU patients makes the specificity of the neurocognitive and behavioral assessment extremely challenging. In the present paper, a multidisciplinary panel of Italian PKU experts discussed different tools available for cognitive, psychopathological, and neurological assessment at different ages based on the existing literature and daily clinical practice. This study aims to provide evidence and a real-life-based framework for a specific clinical assessment of pediatric, adolescent, and adult patients affected by PKU., (© 2022. The Author(s).)

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish., Competing Interests: Competing interests F.C. and S.E.F. have intellectual property rights in the area of hyperspectral imaging and they are co-founders of Kulia Laboratories, a company that is advancing hyperspectral imaging., (© 2022. Published by The Company of Biologists Ltd.)

Adenomatous polyps are precancerous lesions associated with a higher risk of colorectal cancer (CRC). Curcumin and anthocyanins have shown promising CRC-preventive activity in preclinical and epidemiological studies. The objective of this window-of-opportunity, proof-of principle trial was to evaluate the effect of curcumin combined with anthocyanin supplements on tissue biomarkers of colorectal adenomatous polyps. Eligible patients received either anthocyanin and curcumin supplementation or related matching placebo for 4-6 weeks before polyp removal. Adenomatous polyps and adjacent tissue biopsies were collected at baseline and after supplementation for immunohistochemical assessment of β-catenin, NF-kappa B (NF-κB), Ki-67, P53, and dysplasia. No differences were observed in baseline biomarker expression between normal and dysplastic tissues. The combination of anthocyanins and curcumin resulted in a significant borderline reduction of NF-κB immunohistochemistry (IHC) expression in adenoma tissue (geometric mean ratio (GMR): 0.72; 95% confidence interval (CI): 0.51-1.00; p -value: 0.05) and a trend to a reduction of Ki-67 (GMR: 0.73; 95% CI: 0.50-1.08; p -value: 0.11). No significant modulation of biomarkers in normal adjacent mucosa was observed. We concluded that the combined supplementation of anthocyanins and curcumin seems to lead to a potentially favorable modulation of tissue biomarkers of inflammation and proliferation in colon adenomas.

Background: Hyperinsulinemic hypoglycemia (HI) is the most frequent cause of recurrent hypoglycemia in children. Despite diagnostic and therapeutic advances, it remains an important cause of morbidity, leading to neurological complications, such as psychomotor retardation and epilepsy. Patients with diffuse drug-unresponsive HI manifest neurological impairment and neurobehavioral problems, even though surgically treated with a near-total pancreatectomy. Based on the analogies between HI and GLUT1 deficiency, both presenting with neuroglycopenia and lack of alternative cerebral energy sources, we administered a ketogenic diet (KD) in three drug-unresponsive GCK-HI patients with the aim of preserving neurodevelopment and avoiding the need of a near-total pancreatectomy. They presented recurrent symptomatic hypoglycemia, intellectual disability and refractory epilepsy. Patients were treated with classical KD for 79, 27 and 18 months, respectively., Results: All patients became asymptomatic in a few days and showed an important improvement of the alert state. Epilepsy disappeared and no appearance of novel hypoglycemic lesions was detected with a brain MRI. Cognitive and adaptive abilities rapidly improved and normalized. IQ rose significantly from 81 to 111 (p = 0.04) in patient 1, from 82 vs 95 (p = 0.04) in patient 2, from 60 to 90 (p = 0.04) in patient 3., Conclusions: We demonstrated the safety and efficacy of KD in the treatment of drug-unresponsive GCK-HI at a short and long-term. The neuroprotective effects of KD determined the recovery from epilepsy and intellectual disabilities and averted the need of a near-total pancreatectomy. All patients and their families reported an improvement of physical and psychosocial well-being, with a substantial improvement of their quality of life. These results might change the course and the quality of life of these patients and their families, having a relevant impact on human lives. Therefore, KD might be considered the elective treatment in unresponsive forms of GCK-HI., (© 2021. The Author(s).)

Purpose: Low-dose tamoxifen halved recurrence after surgery in a phase III trial in breast noninvasive disease without increasing adverse events. We explored the effect of low-dose tamoxifen in clinically relevant subgroups, including menopausal status, estradiol levels, smoking, body mass index, and proliferation of baseline lesion., Patients and Methods: Incidence of invasive breast cancer or ductal carcinoma in situ was the primary endpoint. HRs and interaction terms were estimated using Cox models., Results: A favorable HR and 95% confidence interval (CI) could be demonstrated for postmenopausal status (HR = 0.30; 95% CI, 0.11-0.82 vs. HR = 0.73; 95% CI, 0.30-1.76 in premenopausal women; P interaction = 0.13), women with estradiol less than 15.8 pg/mL, presence of menopausal symptoms at baseline, and never smoking ( P interaction = 0.07), although the interaction P value was >0.05 for all characteristics. Efficacy was similar in all body mass index categories. Tumors with Ki-67 above the median level of 10% had a greater benefit (HR = 0.27; 95% CI, 0.09-0.81) than those with Ki-67 ≤10% (HR = 1.58; 95% CI, 0.45-5.60; P interaction = 0.04)., Conclusions: The efficacy of low-dose tamoxifen seems to be greater in postmenopausal women and in women with lower estradiol levels. Benefits appear to be larger also in women with menopausal symptoms, never smokers, and tumors with Ki-67 >10%. Our results by menopausal status provide important insight into low-dose tamoxifen personalized treatment, although caution is necessary given their exploratory nature. Observation of an improved response in tumors with Ki-67 >10% is consistent but the use of the marker in this setting is investigational. See related commentary by Fabian, p. 3510 ., (©2021 American Association for Cancer Research.)

Low-dose tamoxifen halves recurrence in non-invasive breast cancer without significant adverse events. Some adjuvant trials with tamoxifen 20 mg/day had shown an association between low endoxifen levels (9-16 nM) and recurrence, but no association with CYP2D6 was shown in the NSABP P1 and P2 prevention trials. We studied the association of CYP2D6 genotype and tamoxifen metabolites with tumor biomarkers and recurrence in a randomized phase III trial of low-dose tamoxifen. Median (IQR) endoxifen levels at year 1 were 8.4 (5.3-11.4) in patients who recurred vs 7.5 (5.1-10.2) in those who did not recur (p = 0.60). Tamoxifen and metabolites significantly decreased C-reactive protein (CRP, p < 0.05), and a CRP increase after 3 years was associated with higher risk of recurrence (HR = 4.37, 95% CI, 1.14-16.73, P = 0.03). In conclusion, endoxifen is below 9 nM in most subjects treated with 5 mg/day despite strong efficacy and there is no association with recurrence, suggesting that the reason for tamoxifen failure is not poor drug metabolism. Trial registration: ClinicalTrials.gov, Identifier: NCT01357772 .

The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.

Purpose: Tamoxifen administered for 5 years at 20 mg/d is effective in breast cancer treatment and prevention, but toxicity has limited its broad use. Biomarker trials showed that 5 mg/d is not inferior to 20 mg/d in decreasing breast cancer proliferation. We hypothesized that a lower dose given for a shorter period could be as effective in preventing recurrence from breast intraepithelial neoplasia but have a lower toxicity than the standard dose., Patients and Methods: We conducted a multicenter randomized trial of tamoxifen, 5 mg/d or placebo administered for 3 years after surgery in women with hormone-sensitive or unknown breast intraepithelial neoplasia, including atypical ductal hyperplasia and lobular or ductal carcinoma in situ. The primary end point was the incidence of invasive breast cancer or ductal carcinoma in situ., Results: Five hundred women 75 years of age or younger were included. After a median follow-up of 5.1 years (interquartile range, 3.9-6.3 years), there were 14 neoplastic events with tamoxifen and 28 with placebo (11.6 v 23.9 per 1,000 person-years; hazard ratio, 0.48; 95% CI, 0.26 to 0.92; P = .02), which resulted in a 5-year number needed to treat of 22 (95% CI, 20 to 27). Tamoxifen decreased contralateral breast events by 75% (three v 12 events; hazard ratio, 0.25; 95% CI, 0.07 to 0.88; P = .02). Patient-reported outcomes were not different between arms except for a slight increase in frequency of daily hot flashes with tamoxifen ( P = .02). There were 12 serious adverse events with tamoxifen and 16 with placebo, including one deep vein thrombosis and one stage I endometrial cancer with tamoxifen and one pulmonary embolism with placebo., Conclusion: Tamoxifen at 5 mg/d for 3 years can halve the recurrence of breast intraepithelial neoplasia with a limited toxicity, which provides a new treatment option in these disorders.

The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.

Background: Epidemiological studies and cardiovascular prevention trials have shown that low-dose aspirin can reduce colorectal cancer (CRC) incidence and mortality, including inhibition of distant metastases. Metformin has also been associated with decreased colon adenoma recurrence in clinical trials and lower CRC incidence and mortality in epidemiological studies in diabetics. While both drugs have been tested as single agents, their combination has not been tested in cancer prevention trials., Methods/design: This is a randomized, placebo-controlled, double-blind, 2 × 2 biomarker trial of aspirin and metformin to test the activity of either agent alone and the potential synergism of their combination on a set of surrogate biomarkers of colorectal carcinogenesis. After surgery, 160 patients with stage I-III CRC are randomly assigned in a four-arm trial to either aspirin (100 mg day), metformin (850 mg bis in die), their combination, or placebo for one year. The primary endpoint biomarker is the change of IHC expression of nuclear factor kappa-B (NFκB) in the unaffected mucosa of proximal and distal colon obtained by multiple biopsies in two paired colonoscopies one year apart. Additional biomarkers will include: 1) the measurement of circulating IL-6, CRP and VEGF; 2) the IHC expression of tissue pS6K, p53, beta-catenin, PI3K; 3) the associations of genetic markers with treatment response as assessed by next generation sequencing of primary tumors; 4) the genomic profile of candidate genes, pathways, and overall genomic patterns in tissue biopsies by genome wide gene expression arrays; and 5) the evaluation of adenoma occurrence at 1 year., Discussion: A favorable biomarker modulation by aspirin and metformin may provide important clues for a subsequent phase III adjuvant trial aimed at preventing second primary cancer, delaying recurrence and improving prognosis in patients with CRC., Trial Registration: EudraCT Number: 2015-004824-77; ClinicalTrial.gov Identifier: NCT03047837 . Registered on February 1, 2017.

Cells use different means to communicate within and between tissues and thereby coordinate their behaviours. Following the initial observations of enigmatic long filopodia unrelated to cell movement, it became clear that the roles of cellular protrusions are not restricted to sensing functions or motility and are much more diverse than previously appreciated. Advances in live-imaging and genetic tools revealed several types of non-conventional cell protrusions and their functions, ranging from tissue patterning, proliferation and differentiation control, tissue matching and cell spacing to more unexpected roles such as priming of cell adhesion as well as bidirectional coordination of tissue movements. Here, we will highlight exciting new insights into highly diverse cell behaviours elicited by protrusions and contact-dependent cell communication, essential for embryonic development across species., (Copyright © 2018. Published by Elsevier Ltd.)

With this survey, we aimed to address the reasons why physicians are reluctant to prescribe breast cancer-preventive therapy with the selective estrogen receptor modulators (SERM) tamoxifen or raloxifene despite a strong evidence of efficacy. A self-administered 5-point Likert questionnaire was given during breast cancer meetings in Europe or sent via email to rank the importance of 10 predefined reasons for low uptake of SERMs for breast cancer therapeutic prevention. Analyses tested the associations between the stated reasons and physician characteristics such as gender, age, country of work, and specialty. Of 246 delivered questionnaires, 27 were incomplete and were excluded from analysis. Overall, there was a small variability in response scores, with a tendency for physicians to give moderate importance (score = 3) to all 10 statements. However, the top five reasons were: the expected greater preventive effectiveness of aromatase inhibitors (70.3% with score >3), difficulty applying current risk models in clinical practice (69.9%), the lack of clarity on the most appropriate physician for prevention advice (68.4%), the lack of reliable short-term biomarkers of effectiveness (67.5%), and the lack of commercial interest in therapeutic prevention (66.0%). The lack of reliable short-term biomarkers showed a tendency to discriminate between medical oncologists and other breast specialists (OR = 2.42; 95% CI, 0.93-6.25). This survey highlights the complexity of prescribing decisions among physicians in this context. Coupled with the known barriers among eligible women, these data may help to identify strategies to increase uptake of breast cancer therapeutic prevention. Cancer Prev Res; 11(1); 38-43. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Positioning organs in the body often requires the movement of multiple tissues, yet the molecular and cellular mechanisms coordinating such movements are largely unknown. Here, we show that bidirectional signaling between EphrinB1 and EphB3b coordinates the movements of the hepatic endoderm and adjacent lateral plate mesoderm (LPM), resulting in asymmetric positioning of the zebrafish liver. EphrinB1 in hepatoblasts regulates directional migration and mediates interactions with the LPM, where EphB3b controls polarity and movement of the LPM. EphB3b in the LPM concomitantly repels hepatoblasts to move leftward into the liver bud. Cellular protrusions controlled by Eph/Ephrin signaling mediate hepatoblast motility and long-distance cell-cell contacts with the LPM beyond immediate tissue interfaces. Mechanistically, intracellular EphrinB1 domains mediate EphB3b-independent hepatoblast extension formation, while EpB3b interactions cause their destabilization. We propose that bidirectional short- and long-distance cell interactions between epithelial and mesenchyme-like tissues coordinate liver bud formation and laterality via cell repulsion., (Copyright © 2016. Published by Elsevier Inc.)

A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells.

Background: Congenital hyperinsulinism (CHI) is the most frequent cause of hypoglycemia in children. In addition to increased peripheral glucose utilization, dysregulated insulin secretion induces profound hypoglycemia and neuroglycopenia by inhibiting glycogenolysis, gluconeogenesis and lipolysis. This results in the shortage of all cerebral energy substrates (glucose, lactate and ketones), and can lead to severe neurological sequelae. Patients with CHI unresponsive to medical treatment can be subjected to near-total pancreatectomy with increased risk of secondary diabetes. Ketogenic diet (KD), by reproducing a fasting-like condition in which body fuel mainly derives from beta-oxidation, is intended to provide alternative cerebral substrates such ketone bodies. We took advantage of known protective effect of KD on neuronal damage associated with GLUT1 deficiency, a disorder of impaired glucose transport across the blood-brain barrier, and administered KD in a patient with drug-unresponsive CHI, with the aim of providing to neurons an energy source alternative to glucose., Methods: A child with drug-resistant, long-standing CHI caused by a spontaneous GCK activating mutation (p.Val455Met) suffered from epilepsy and showed neurodevelopmental abnormalities. After attempting various therapeutic regimes without success, near-total pancreatectomy was suggested to parents, who asked for other options. Therefore, we proposed KD in combination with insulin-suppressing drugs., Results: We administered KD for 2 years. Soon after the first six months, the patient was free of epileptic crises, presented normalization of EEG, and showed a marked recover in psychological development and quality of life., Conclusions: KD could represent an effective treatment to support brain function in selected cases of CHI.

Background: Twenty-five patients with Niemann Pick disease type C (age range: 7 months to 44 years) were enrolled in an Italian independent multicenter trial and treated with miglustat for periods from 48 to 96 months., Methods: Based on the age at onset of neurological manifestations patients' phenotypes were classified as: adult (n = 6), juvenile (n = 9), late infantile (n = 6), early infantile (n = 2). Two patients had an exclusively visceral phenotype. We clinically evaluated patients' neurological involvement, giving a score of severity ranging from 0 (best) to 3 (worst) for gait abnormalities, dystonia, dysmetria, dysarthria, and developmental delay/cognitive impairment, and from 0 to 4 for dysphagia. We calculated a mean composite severity score transforming the original scores proportionally to range from 0 to 1 to summarize the clinical picture of patients and monitor their clinical course., Results: We compared the results after 24 months of treatment in 23 patients showing neurological manifestations. Stabilization or improvement of all parameters was observed in the majority of patients. With the exception of developmental delay/cognitive impairment, these results persisted after 48-96 months in 41 - 55% of the patients (dystonia: 55%, dysarthria: 50%, gait abnormalities: 43%, dysmetria: 41%, respectively). After 24 months of therapy the majority of the evaluable patients (n = 20), demonstrated a stabilization or improvement in the ability to swallow four substances of different consistency (water: 65%, purée: 58%, little pasta: 60%, biscuit: 55%). These results persisted after 48-96 months in 40-50% of patients, with the exception of water swallowing. Stabilization or improvement of the composite severity score was detected in the majority (57%) of 7 patients who were treated early (within 3.5 years from onset) and rarely in patients who received treatment later., Conclusions: The results of this study suggest that miglustat treatment can improve or stabilize neurological manifestations, at least for a period of time; the severity of clinical conditions at the beginning of treatment can influence the rate of disease progression. This conclusion applies particularly to patients with juvenile or adult onset of the disease., Trial Registration: EudraCT number 2006-005842-35.

Organs like the vertebrate vascular system and the insect tracheal system develop from separate primordia that undergo fusion events to form interconnected tubular networks. Although the correct pattern of tubular connections (anastomoses) in these organs is crucial for their normal function, the cellular and molecular mechanisms that govern tube fusion are only beginning to be understood. The process of tube fusion involves tip cell specification, cell-cell recognition and contact formation, self-avoidance, changes in cell shape and topology, lumen formation, and luminal membrane fusion. Significant insights into the underlying cellular machinery have been provided by genetic studies of tracheal tube fusion in Drosophila. Here, we summarize these findings and we highlight similarities and differences between tube fusion processes in the Drosophila tracheae and in the vertebrate vascular system. We integrate the findings from studies in vivo with the important mechanistic insights that have been gained from the analysis of tubulogenesis in cultured cells to propose a mechanistic model of tube fusion, aspects of which are likely to apply to diverse organs and organisms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The primary aim of this study was to assess cognitive development, in particular that of executive functions (EFs), and behavioral findings for patients with early treated phenylketonuria (PKU). Furthermore, we evaluated the relationships of our findings with plasma levels of Phe and adherence to dietary prescriptions., Methods: A cross-sectional design was adopted. Patients who had early treated PKU, who were older than 4 years, and who were regularly seen by a physician were enrolled in the study. Cognitive development was assessed with Wechsler Scales appropriate to the chronological age of subjects in the study. Executive functions were assessed using the Tower of London test, and behavioral findings were quantified with the Child Behavior Checklist., Results: Thirty-five patients were enrolled (mean age 11.5 years, SD ± 6.2). The mean Full Scale intelligence quotient was in the normal range (93.4 ± 17.4), without significant difference between the verbal intelligence quotient (mean, 94.3 ± 16.1) and performance intelligence quotient (mean, 93.9 ± 18.0). The majority of patients showed a deficit within the EF domain of cognitive abilities (mean lower than 1.8 SD of the normal mean), 8 of them showing a score <2 SD lower than the normal mean. Internalizing problems were also observed in 12 patients (38.7%) and were higher in adherent patients and in patients with a lower intelligence quotient., Conclusion: After early diagnosis and treatment of PKU, residual problems can be found in EFs of patients not achieving satisfactory Phe levels, while scores of internalizing behaviors were higher in compliant patients.

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. Here, we show that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. We demonstrate that Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network.

Tumor progression is a key aspect in oncology. Not even the overexpression of a powerful oncogenic stimulus such as platelet derived growth factor-B (PDGF-B) is sufficient per se to confer full malignancy to cells. In previous studies we showed that neural progenitors overexpressing PDGF-B need to undergo progression to acquire the capability to give rise to secondary tumor following transplant. By comparing the expression profile of PDGF-expressing cells before and after progression, we found that progressed tumors consistently downregulate the expression of the antiproliferative gene Btg2. We therefore tested whether the downregulation of Btg2 is sufficient and necessary for glioma progression with loss and gain of function experiments. Our results show that downregulation of Btg2 is not sufficient but is necessary for tumor progression since the re-introduction of Btg2 in fully progressed tumors dramatically impairs their gliomagenic potential. These results suggest an important role of Btg2 in glioma progression. Accordingly with this view, the analysis of public datasets of human gliomas showed that reduced level of Btg2 expression correlates with a significantly worse prognosis.